Project Summary Vaping? electronic cigarettes (e-cigs) is believed to be less toxic than smoking tobacco, leading the physician groups such as the Royal College of Physicians to recommended e-cig use as a way to quit tobacco smoking in a risk reduction approach. Such advice, however, may be misinterpreted to imply that vaping has no ill effects upon health, especially for previous never-smokers. In support of this notion, an astonishing 11% percent of never smoking high school students admit to consistent vaping. Unfortunately, there is growing evidence of harm from vaping, including the development of chronic bronchitis. Thus, the potential damage of vaping to never smokers, especially the vulnerable adolescent population, needs to be explored. To this end, this application examines how e-cigarette vapors adversely impact human airways. Data need to establish which e-cig components are most harmful ? nicotine, the vaping fluid, or added synthetic flavors ? and how they mechanistically exert their effects at the primary target site of inhalation, namely the airway epithelium. To answer these questions, we assembled a multidisciplinary team with expertise in airway biology to study the extent by which e-cig vapor affects airway health. We will investigate the effect of e-cig vapor on airway epithelia in vitro (aim 1), on mucociliary function in a new ovine, large animal model (aim 2), and in a cross- sectional cohort of young never smokers who started to vape (aim 3). The latter group of e-cig users is at greatest risk for harm. Aim 1 will mechanistically examine the signaling pathway of e-cig vapor at the airway epithelium, triggering TRP receptors to stimulate TGF-b1 and Notch signaling to cause mucociliary dysfunction, a hallmark of chronic bronchitis. This aim will be accomplished by exposing fully differentiated airway epithelial cells in vitro to e-cig vapor and its components using sophisticated exposure robots. Aim 2 will confirm the mechanisms studied in vitro in a new, ovine large animal model that allows to examine the effects of vapor components on parameters of mucociliary function and investigate airway inflammation. Finally, aim 3 will examine parameters of mucociliary dysfunction and airway inflammation in young never smokers who started to vape for at least 6 months using a cross sectional design. History of vaping and topography will be assessed in objective ways to correlate with outcomes. This application will therefore mechanistically examine harmful effects of e-cig vapor on airway function and inflammation in a team science approach, going from bench to animal model to human subjects.